The expression of chemokines within tissues regulates the recruitment of specific subsets of lymphocytes to distinct tissue sites. Chemokines are therefore responsible, in part, for directing the immune response that ensues following bacterial invasion. Our results are the first to demonstrate that there are regional differences in chemokine expression within the female reproductive tract in response to Chlamydia
infection. Although homeostatic differences in CCL25 (thymus-expressed chemokine) expression have been demonstrated within regions of the intestinal tract (19
), this is the first report demonstrating regional chemokine differences in response to infection. Studies measuring cellular influx and adhesion molecule expression during Chlamydia
infection first suggested that there were regional differences in the immune response between the cervical-vaginal region and oviducts of mice (18
). Our data further support this theory, as we found that chemokines associated with Th1 responses were present at significantly higher levels in the oviducts than in the cervical-vaginal tissues of mice during infection. Namely, CXCL10 (IP-10) and CXCL9 (MIG) protein levels peaked early in infection in the upper GT and then returned to the baseline, whereas the CCL5 (RANTES) level remained elevated for the duration of infection. These results were confirmed at the mRNA level, although CXCL9 (MIG) but not CXCL10 (IP-10) mRNA levels were significantly higher in the upper GT than in the lower GT. This finding may have been due to the fact that the CXCL10 (IP-10) protein level in the upper GT peaked at a time point earlier than the one at which we measured mRNA. Although we have not yet confirmed the functions of these chemokines, there are data to suggest that the concentrations reached during infection are sufficient for lymphocyte recruitment (29
). Experiments are under way to demonstrate that the induction of CXCL9 (MIG), CXCL10 (IP-10), and/or CCL5 (RANTES) is responsible for the selective recruitment of Th1 cells to the upper GT during infection.
Compared to the results for the upper GT, the chemokine expression patterns differed quantitatively and kinetically in the cervical-vaginal region during infection. First, only low levels of Th1-associated chemokines were present in the lower GT. Second, CCL11 (eotaxin) levels were significantly increased late in the course of infection. Immunohistochemical staining supported these findings by showing that CCL11 (eotaxin) expression was confined to epithelial cells during the resolution phase of infection (day 21). However, the mRNA expression of CCL11 (eotaxin) increased in the lower GT relative to the upper GT early after infection but not at later time points, when the expression of CCL11 (eotaxin) protein was significantly elevated. Although mice cleared infection in the lower GT, the diminished production of Th1-associated chemokines in that region may have been responsible for the reduced numbers of CD4 cells observed in the lower GT. It is possible that ascending infection correlates with smaller numbers of CD4 Th1 cells in the lower GT.
Considering the anatomical and functional differences between the oviducts and cervical regions of the GT, it is not unanticipated to find immunologically distinct responses at these sites. For instance, epithelial cells are different at the two sites. Squamous epithelial cells are found in the cervical region, while ciliated columnar epithelial cells line the oviducts. Epithelial cells play a central role in directing the immune response, since they host Chlamydia
and secrete cytokines, such as IL-8, early after infection (27
). Moreover, endocervical but not endometrial cell lines secrete IL-8 in response to Chlamydia
), suggesting that epithelial cells at these discrete sites respond differently to infection. In addition, we found that CXCL10 (IP-10) was expressed on a wider array of cell types in the upper GT than in the lower GT, further supporting the concept that chemokine secretion differs between the upper and lower GTs.
The differences in chemokine expression in the upper and lower GTs cannot be explained by simple differences in the level of infection between these two regions. Our data show that the level of infection was significantly higher in the lower GT early in infection, at a time when the levels of Th1-associated chemokines were significantly higher in the upper GT. Likewise, chlamydia levels were similar in the upper and lower GTs during the second week of infection, although resolution of infection occurred more quickly in the lower GT. By day 35, the lower GT was negative for chlamydiae, while the upper GT was either negative or had minimal numbers of inclusions. These results are similar to those previously reported (18
), although our data indicate more variability in the numbers of chlamydiae detected in the upper and lower GTs throughout infection and suggest that there may be a small lag in the clearance of chlamydiae from the upper GT. Also, to rule out the possible influence of inoculating dose on chemokine levels, we found no differences in the levels of CXCR10 (IP-10) and CCL11 (eotaxin) in mice infected with 1.5 × 105
IFU of MoPn (data not shown). These data, coupled with the results of the immunohistochemical analysis showing that chemokine expression occurs in noninflammatory cell types early after infection, suggest that chemokine expression in the upper GT precedes the recruitment of inflammatory cells and is not influenced by the inoculating dose. These conclusions are not surprising, since all somatic cells produce chemokines and in other models, chemokine production has been shown to precede the influx of inflammatory cells (20
Our results showing that steady, basal levels of CXCL10 (IP-10) and CCL11 (eotaxin) are maintained in the upper and lower GTs of uninfected mice treated with medroxyprogesterone acetate indicate that the chemokine differences seen between the upper and lower GTs of infected mice are not due to progesterone treatment. Female reproductive hormones have been reported to alter cytokine production (10
), and other data have shown that the expression of some chemokines varies with hormonal fluctuations during normal menstruation. For example, increased immunoreactivity to CCL11 (eotaxin) has been observed in endometrial epithelium during the luteal phase (high progesterone) of the mouse menstrual cycle compared to the follicular phase (low progesterone) (13
). In contrast, Saavedra and colleagues (28
) reported that estrogen treatment did not alter CCL2 (MCP-1), Mip-2, or CCL5 (RANTES) levels over a 21-day period. In our model, progesterone treatment did not appear to influence CXCL10 (IP-10) and CCL11 (eotaxin) levels, verifying that the increases observed were produced in response to infection.
CCL5 (RANTES) was the only chemokine to stay at significantly elevated levels in the upper GT throughout the course of infection. However, mRNA expression was low on all days that were evaluated. The presence of CCL5 (RANTES) protein in tissue has generally been shown to correlate with mRNA expression, making our results somewhat surprising. A possible explanation is that CCL5 (RANTES) protein is delivered to the tissue from other sites. CCL5 (RANTES) is found at picogram levels in the blood of healthy humans (8
) and is known to be released from thrombin-stimulated platelets (16
). It is therefore possible that the CCL5 (RANTES) protein measured in the GT following Chlamydia
infection is blood derived rather than locally produced. Upon secretion, CCL5 (RANTES) may then directly bind to the activated genital endothelium (34
SuperArray analysis allowed the evaluation of additional chemokines in the local genital mucosa of infected mice. Other chemokines detected by this analysis include CCL21 (SLC), Gro-1, TCA-3, XCL1 (lymphotactin), CXCL4 (platelet factor 4), CXCL12 (SDF-1α/β), and SDF-2. Most notably, there was an increase in the level of CCL21 (SLC) mRNA in infected upper GT tissues compared to uninfected tissues. CCL21 (SLC) is important for T-cell migration across high endothelial venules within secondary lymphoid tissues, as demonstrated for mice deficient in CCL21 (SLC) (11
) or the chemokine receptor CCR7 (38
). However, CCL21 (SLC) has also been shown to bind to CXCR3, the receptor for CXCL9 (MIG) and CXCL10 (IP-10) in mice but not humans (31
). Preliminary data obtained with reverse transcription-PCR for whole GT homogenates have shown that CXCR3 and CCR5 are expressed only in the GTs of infected mice and not until 14 days after infection (unpublished observations). In addition, we found that the levels of CXCL12 (SDF-1α/β) mRNA expression were similar between infected and uninfected tissues but were approximately twofold higher in upper GT tissue than in lower GT tissue (data not shown). CXCL12 (SDF-1α/β) induces rapid adhesion of CD4 cells to CD54 (5
). Thus, CXCL10 (IP-10), CXCL9 (MIG), CCL5 (RANTES), CCL21 (SLC), and CXCL12 (SDF-1α/β) are most likely involved in the chemotaxis of Th1 cells to the upper GT during Chlamydia
To date, there have been very few reports of chemokine induction in response to Chlamydia
infection in the GT. Previous reports have examined chemokine induction in vitro and have focused on chemokines of the CXC class, which are important for neutrophil chemotaxis. Namely, IL-8 (27
), CXCL1 (Gro-α), and CXCL5 (epithelial neutrophil activating protein 78 [ENA-78]) (35
) were produced by epithelial cells infected with human serovars of C
. Interestingly, IL-8 was not found in vaginal secretions of women with C
). However, Mip-2 and CCL2 (MCP-1) were found at increased levels in the lungs of mice during infection with Chlamydia psittaci
). In this study, we noted an increase in Gro-1 but not Mip-2, the functional homolog of murine IL-8. We also found that CCL2 (MCP-1) mRNA expression was consistently low in both the upper and the lower GTs, supporting our protein data. CCL2 (MCP-1) has been shown to be upregulated in vaginal tissues of mice following infection with Candida albicans
in vivo (28
). These differences in Mip-2 and CCL2 (MCP-1) expression may reflect differences between tissue sites or specific features of the pathogens.
The factors that lead to ascending Chlamydia
infection in a subset of individuals are currently unknown. Our data showing differential chemokine expression in the upper and lower GTs support increasing evidence that the inflammatory response in the lower GT may be prematurely terminated even in the presence of an active C
infection. Perhaps Chlamydia
-infected cells secrete immunosuppressive factors which hamper antichlamydial immunity in the lower GT. Alternatively, early termination of inflammatory responses in the lower GT may be an inherent response of a site that is commonly exposed to nonpathogenic organisms. For example, using another mucosal tissue that is exposed to commensal flora, Yamamoto et al. showed that intestinal epithelial cells inhibit T-cell responses through a novel, non-transforming growth factor β-dependent mechanism (36
). Interestingly, the early production of gamma interferon (6
) and tumor necrosis factor α (7
) in vaginal secretions and the expression of adhesion molecules in the lower GT early after infection (18
) diminished to nearly baseline levels by day 7 in the presence of viable chlamydiae. Therefore, we hypothesize that delayed eradication of chlamydiae in the lower GT early after infection may facilitate upper GT infection. Future studies will therefore be aimed at selectively boosting the antichlamydial immune response in the cervical-vaginal region.